Ubiquitin-proteasome systems and their homologs are attractive targets for the treatment of some of the most formidable public health challenges of this century including cancer, viral infections, tuberculosis, hypercholesteremia and neurodegeneration. Promising news is that use of proteasome inhibitors, in combination with immunomodulatory drugs, has greatly extended the lifespan of patients with relapsed/refractory multiple myeloma. The fundamental knowledge that enabled the development of these inhibitors was spearheaded by structure-function studies of proteasomes from Archaea. Recently, we discovered that Archaea synthesize ubiquitin-like proteins named SAMPs that are attached by isopeptide bonds to lysine residues of target proteins by an E1-like mechanism (sampylation) that resembles ubiquitylation. SAMPs also mobilize sulfur to form thiolated tRNA and molybdopterin. We hypothesize that SAMPs provide a window for understanding how ubiquitin-like proteins evolved to control cell function including the transient inactivation of enzyme function, targeting of proteins for destruction by proteasomes and coordination of protein modification with sulfur mobilization pathways associated with stress responses. In our research, we demonstrate that polymeric chains of SAMPs are attached near the conserved active site residues of thiouridine synthetase (NcsA), an enzyme linked to oxidative and thermal stress and associated with ubiquitin- like proteins in all domains of life. We also find that many of the lysine residues modified by `sampylation' are near catalytic active sites suggesting a general mechanism of transient enzyme inactivation that may be conserved in eukaryotic cells. SAMPs are also found to target proteins for destruction by mechanisms that appear to require N-terminal degrons and 26S proteasome components. Here we will use the halophilic archaeon Haloferax volcanii as a model to provide insight into how ancient ubiquitin-like modification pathways may control biological activity. In Aim 1, we will define and determine the biological role of the formation of polymeric ubiquitin-like chains of SAMP2 on the thiouridine synthetase (NcsA) associated with maintaining translation fidelity and overcoming thermal stress. In Aim 2, we will provide mechanistic insight into SAMP attachment at or near catalytic active site residues to infer general principles of enzyme regulation that may extend across domains of life. In Aim 3, we will define the interactions of the SAMPs with proteasome-associated AAA ATPases and JAMM/MPN+ domain proteins that may be conserved through evolution. At the conclusion of these studies, we will have expanded our knowledge of ubiquitin-like protein modification and provided an evolutionary perspective on how these attachments may regulate enzyme activity, proteolysis and association with protein partners including proteasomes.